Ras and related small molecular weight GTPases function in the regulation of signaling and cell growth, and collectively serve to control cell proliferation, differentiation and apoptosis (FIG. 1) [Takai et al. 2001; Wennerberg et al. 2005]. The Ras-related GTPases are divided into four subfamilies with the Rab proteins regulating membrane transport, Rho proteins (including Rac and Cdc 42) regulating cytoskeletal rearrangements and responses to signaling, Arf/Sar proteins regulating membrane and microtubule dynamics as well as protein transport, and Ran proteins controlling nucleocytoplasmic transport.
Ras and Ras-related GTPase functions are tightly regulated (FIG. 2), and dysregulation is causal in a wide variety of human diseases. Ras mutations resulting in impaired GTP hydrolysis and plasma membrane hyperactivation are linked to many human cancers [Farnsworth et al. 1991; Sukumar et al. 1983; Taparowsky et al. 1982; Boylan et al. 1990; Hruban et al. 2004; Abrams et al. 1996]. Point mutations in the Rab and Rho GTPases are also causal in diverse human diseases affecting pigmentation, immune, and neurologic functions [Houlden et al. 2004; Verhoeven et al 2003; Williams et al. 2000; Bahadoran et al. 2003; and preliminary findings]. Rab and Rho mutants identified in human disease act as dominant negatives either due to a failure to bind GTP or due to inappropriate coupling of the active proteins with downstream effectors. To date, inhibition of Ras and Ras-related proteins has relied largely on altering membrane recruitment with various drugs affecting prenylation [Morgillo F Lee H Y, 2006; Russell R G, 2006; Park, et al. 2002]. Generally, Ras proteins must be farnesylated for proper membrane localization, while Rab and Rho proteins are geranylated. Such strategies lack specificity and are problematic because each of these prenylation machineries is required for the proper function of many Ras superfamily members. Rational drug design has only recently been applied to identify the first two small molecule inhibitors of Rho GTPase family members [Gao, et al. 2004; Nassar et al. 2006]. There are currently no nucleotide binding inhibitors or analogs for any member of the GTPase superfamily, prompting us to undertake a high throughput screen resulting in the identification of novel chemical entitites described herein (FIG. 3-4).
The Rab GTPase subfamily is responsible for regulating membrane transport of proteins and lipids shuttling between various intracellular destinations with individual members governing specific transport events and causal in human disease (FIG. 5-6). Rab7 is a regulator of transport from early to late endosomes and as such is critical for growth factor receptor down-regulation, for control of cell fate through autophagy pathways, nutrient uptake, immune cell regulation, to name a few (FIG. 7). Thus, Rab7 is a late endosome-/lysosome-associated small GTPase. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome (or LRO (lysosome-related organelle)) and phagocytosis. These processes are important in substrate degradation, antigen presentation, cell signaling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neurologic disorders (Alzheimer's, Downs, sensory neuropathies), bone metabolic disorders, cancer and lipid metabolism or storage diseases (FIGS. 6, 19-20). Rab7 inactivation plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses {Zhang, M., Cheng, L., Wang, S, and Wang, T. Biosci Rep 2009, 29:193-209).
Hereditary sensory neuropathies (HSNs) are a group of genetically determined peripheral neuropathies with prominent disturbance of the peripheral sensory neurons (FIG. 19). They are characterized by sensory loss, insensitivity to pain, a variable degree of muscle weakness and wasting, as well as autonomic features. Frequent complications are foot ulcerations and infections that may lead to osteomyelitis, followed by necrosis and amputations. Consequently, the hereditary sensory neuropathies have also been termed ulceromutilating neuropathies. On the other hand, in the presence of additional motor weakness, they have been sub-classified among the group of Charcot-Marie-Tooth (CMT) disorders, an autosomal dominant inherited disorder (˜1:2500) that causes peripheral neuropathy, foot ulcers and frequently requires amputation. Sporadic and familial cases with different modes of inheritance are known to affect both children and adults. The most prevalent forms of the autosomal dominantly inherited hereditary sensory neuropathies are HSN I and CMT2B. HSN1 is associated with mutations in the SPTLC1 gene, whereas mutations in the Rab7 gene have been identified in CMT2B that result in mutant proteins with single amino acid substitutions of highly conserved residues (FIG. 20). The mutants are thought to disrupt critical functions of Rab7 in shuttling cargo between the neuronal synapse and the soma where signaling to the nucleus occurs. The enumerated roles and disease associations of Rab7 thus provides a rationale to identify small molecules that may functionally modulate Rab7 GTPase. Currently, there are no small molecules directed against any member of the Rab GTPase family despite their diverse cellular funtions that ensure normal physiology.